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vol. 168, supplement
the american naturalist
december 2006
Exploring Evolutionary Constraints Is a Task for
an Integrative Evolutionary Biology
P. M. Brakefield* and J. C. Roskam
Institute of Biology, Leiden University, P.O. Box 9516, 2300 RA
Leiden, The Netherlands
abstract: Judging by the volume of writings about evolutionary
constraints, they are an important topic in evolutionary biology.
However, their involvement in shaping patterns of evolutionary
change from morphological stasis to adaptive radiation remains contentious. This is at least in part because of the paucity of robust
analyses of potential examples of constraints, whether of a more
absolute or a relative nature. Here, we argue that what is needed to
explore the type of constraints and bias on evolutionary change that
may emerge from the way in which phenotypic variation is generated
is an integrative approach applied to systems that can be tackled at
different levels of biological organization. This is illustrated using
research on the evolution of patterns in butterfly wing eyespots that
has applied a combination of evolutionary genetics and evo-devo to
an emerging model species with the beginnings of a comparative
approach to describe patterns of variability among the extant taxa
of two species-rich genera.
Keywords: butterfly eyespots, developmental bias, natural selection,
morphospace.
An integrative approach that combines evolutionary genetics and evo-devo in a comparative framework can begin
to address certain issues about morphological evolution
that have always been of great interest but have, until
recently, been beyond the scope of direct experimental
analysis. We will illustrate this approach by using the background and results of one particular experiment with butterfly wing eyespots (Beldade et al. 2002b), together with
some new insights from extending the work to include a
more comparative framework. The main issue is the extent
to which patterns of phenotypic change in adaptive evolution are shaped not only by natural selection but also
by processes that underlie the generation of phenotypic
variation and that could result in what can be called, col* Corresponding author; e-mail: [email protected].
Am. Nat. 2006. Vol. 168, pp. S4–S13. 䉷 2006 by The University of Chicago.
0003-0147/2006/1680S6-41372$15.00. All rights reserved.
lectively, generative constraints. In other words, what are
the roles of such generative constraints in influencing what
actually happens in evolution and in contributing to the
patterns of how species occupy morphological space?
Bridging different levels of biological organization can
also more effectively make the links from genes to evolutionarily relevant phenotypes to variation in fitness in natural populations. By placing this type of integrative analysis
of adaptive evolution that is now becoming possible in many
emerging model organisms within a comparative framework, we can then explore open issues, including the role
of generative constraints. In addition, an ability to examine
not only adaptive evolution but also the processes of speciation and phylogeography within such systems will provide rich data on the extent to which evolutionary change
involving adaptive responses to novel environments is associated with the processes of speciation.
Spectacular patterns of adaptive radiation among species
of a lineage have always enthralled biologists, but even in
such cases, it remains unclear how all-powerful natural
selection is. Studies of evolutionary ecology and measurements of natural selection in examples such as the Galapagos finches (Grant and Grant 2002), anolis lizards (Losos
et al. 2004; Harmon et al. 2005), sticklebacks (Schluter
2000), and the hummingbird-Heliconia system (Temeles
and Kress 2003) readily steer biologists toward the perception that natural selection is highly dominant, at least
when vacant ecological niches and relaxed competition are
involved. However, an argument for taking a more openminded position can be made. This is illustrated most
forcefully in the case of the species flocks of cichlid fishes
in lakes of the African Rift Valley (Fryer and Iles 1972;
Kocher et al. 1993; Salzburger and Meyer 2004; Albertson
and Kocher 2006).
Independent radiations of the cichlids in these lakes have
each led to a series of highly divergent trophic morphs in
which whole suites of morphological, life-history, and behavioral traits have changed. This is dramatic enough within
each lake, but what is even more striking is the degree of
convergence in the trophic morphs in different lakes; the
same modes of coordinated traits are apparent. Is such con-
Exploring Evolutionary Constraints
vergence accountable purely on the basis of closely comparable environmental and ecological opportunities leading
to intense selection for certain directions of change, or are
there properties of the genetical and developmental systems
that underlie the suites of traits that introduce bias and thus
lend themselves to evolution in the observed directions? In
the latter case, the details of how the different traits eventually distill as emergent entities to produce the observed
variability around parallel series of trophic morphs represent
some sort of compromise between how the environments
are dictating natural selection and the processes that generate the phenotypic variation to be sieved by such selection.
Perhaps, if the latter processes were completely flexible and
in no way limiting in either a relative or an absolute manner,
the phenotypes that evolved would not be precisely those
present in the lakes today and would not be so similar in
the different lakes.
Such hypotheses need to be tested in specific cases before any robust assessment is possible of the extent of any
compromise between extrinsic and intrinsic factors in
shaping patterns of evolution, whether involving change
or stasis (see Gould and Lewontin 1979; Maynard Smith
et al. 1985; Antonovics and van Tienderen 1991; Schwenk
1995; Schlichting and Pigliucci 1998; Gould 2002; Stearns
2002; Brakefield 2003, 2006; Blows and Hoffmann 2005;
Eldredge et al. 2005). Perhaps natural selection is allpowerful but further experimental work is necessary on
systems that lend themselves to examining the processes
that generate phenotypic variation in the context of the
ecological arena of natural selection before such a conclusion can be reached. Description of patterns of divergence among taxa within some multidimensional representation of trait space, even in the light of measurements
of fitness curves, is not sufficient to exclude possible
sources of bias on evolution that are introduced through
either genetic variance-covariances or developmental
mechanisms. Experimental tests are also needed in combination with an evo-devo understanding of the processes
that map phenotypes onto genotypes (Beldade and Brakefield 2003b; Brakefield et al. 2003; Fuller et al. 2005; and
see Frankino et al. 2005, 2006).
With respect to genetic variation, lineages may tend to
evolve in patterns that follow genetic lines of least resistance when responding to environmental change or ecological opportunities (Schluter 1996). In developmental
terms, the way in which the phenotype is made or in which
variation in the phenotype maps onto the genetic variances
may also bias or channel evolution in particular directions
(Maynard Smith et al. 1985). Here, the emphasis is not
on absolute constraints, on what could never be built even
with unlimited input from mutation or recombination,
but rather on relative constraints by which some directions
of evolution are more likely to occur than others. If natural
S5
selection in a direction away from a genetic line of least
resistance or a pattern of developmental bias is not sufficiently intense, stable, or prolonged, then evolution in
that direction may not occur.
This is the type of issue we are exploring by using an
integrative approach in tropical butterflies of the speciesrich genus Bicyclus (Condamin 1973). In particular, we
use artificial selection experiments in our laboratory model
species, Bicyclus anynana, to ask whether, for sets of traits
with a common genetic basis or a shared developmental
pathway, the properties of generating the phenotype can
become reflected in patterns of diversity. We work with
different classes of traits in B. anynana (Wijngaarden et
al. 2002; Zijlstra et al. 2002, 2004; Frankino et al. 2005,
2006; Fischer et al. 2006). Studies have included those on
the evolution of the scaling of wing size to overall body
size or of forewing size to hindwing size. We were able to
begin to distinguish between alternative explanations for
the low phenotypic variability associated with such allometric relationships in terms of either developmental constraints or stabilizing natural selection (Frankino et al.
2005, 2006).
Here, we will focus on the wing eyespot pattern. Artificial
selection is applied to pairs of such interacting traits to
examine how they can evolve in the short term in different
directions through trait or morph space. The responses in
our experiments must be largely accounted for by the properties of standing genetic variation in the founder stock
(contrast with long-term experimental evolution in replicated populations of E. coli; Cooper et al. 2003; Elena and
Lenski 2003). The phenotypes that result from artificial selection can also be used to examine their genetic basis as
well as how developmental mechanisms have been modulated during evolution. In addition, we can now begin to
explore the extent to which our understanding of the shortterm evolution of correlated traits in experimental populations of B. anynana can inform us about the patterns of
phenotypic variation among taxa within this genus.
The Eyespot Pattern of the Butterfly Bicyclus anynana
The margins of the wings of many butterflies, including
Bicyclus anynana, are patterned with series of small eyespots. Each eyespot in B. anynana consists of a white central pupil surrounded by two or three concentric rings
with a brown, black, or gold color. Each eyespot ring comprises many hundreds of individual epithelial scale cells
that during development all become fated to synthesize a
particular color pigment. The functional effectiveness of
each eyespot is dependent on the process by which the
different developmental fates of these scale cells in different
color rings are specified.
The eyespot phenotype is important in a functional con-
S6 The American Naturalist
text for Bicyclus butterflies. We know this from field biology and capture-recapture experiments in Malawi for
the ventral wing surfaces that are exposed to potential
predators in butterflies that are at rest with their wings
closed (Brakefield and Frankino 2006). The eyespots on
the ventral wing surfaces show striking phenotypic plasticity in the form of seasonal polyphenism (Brakefield et
al. 1996). The relative fitnesses of butterflies with different
eyespot patterns depend on the seasonal environments in
which they have to evade predators and reproduce. Marginal eyespots are thought to function in butterflies at rest
by deflecting the attacks of vertebrate predators away from
the vulnerable body toward the eyespot “targets”; the margins of the wings tear away easily, enabling some butterflies
at least to escape even after being grabbed by a bird or
lizard. This hypothesis has proved difficult to evaluate, but
the most recent results provide some support for it, at least
when the predators concerned are naive birds (Lyytinen
et al. 2003, 2004). The function of the eyespots on the
dorsal surfaces of the wings has been unclear because they
are not exposed to potential predators in resting butterflies.
However, recent work is beginning to implicate them in
mate choice and sexual selection and perhaps in species
recognition (Breuker and Brakefield 2002; Robertson and
Monteiro 2005).
Shared Genetics and Development of Eyespots
The eyespots on the wings of many butterflies and moths
are ideal for exploring the flexibility of morphological
change in different directions within trait space for patterns or modules made up of serial repeats of the same
basic element (Brakefield 2003). The evolution of mammalian dentition and of the subsets of teeth with divergent
morphologies provide further examples of this type of
modular pattern for which exciting advances are being
made in understanding the genetic and developmental basis of divergent morphologies both within and among species and also, in this case, in the fossil record (SalazarCiudad and Jernvall 2002; Kangas et al. 2004).
Many primitive species of Lepidoptera (as well as more
derived species) have a series of small undifferentiated dark
spots evenly spaced along their wing margins. Each spot is
positioned along an internervule between pairs of the major
proximal-distal wing veins that divide the wing into socalled wing cells. This probably reflects a phenotypic module
comparable with the early novelty that saw the origin of the
developmental capacity to make patterns of repeated spots
in each wing cell. In the Nymphalidae, and probably in
several other lineages of higher Lepidoptera, such a simple
pattern has become much elaborated to yield series of repeated marginal eyespots, each with concentric rings of
color. The eyespots can vary in both size and color pattern,
and the processes that underlie the gain in eyespot individuality and pattern complexity are the focus of our research.
Thus, an initially simple module of repeated spots has been
elaborated in evolution to yield a spectacular diversity of
patterns across taxa that is associated with an increasingly
hierarchical structure for the whole module (Brunetti et al.
2001; Beldade et al. 2002c; Arbesman et al. 2003; Monteiro
et al. 2003). This type of process can also be expressed in
the context of an increasing evolvability of the whole module
through time. How is this achieved? In addition, what are
the consequences of such a process in terms of the occupation of morphological space and of generative constraints
on future evolutionary change?
The results of evo-devo work on Bicyclus anynana are
consistent with this general scenario. It is clear that all
eyespots in this species, as well as in other members of
the Nymphalidae, are formed by the same developmental
pathway that is part of wing development in late larvae
and early pupae (Brunetti et al. 2001; Beldade and Brakefield 2002; Reed and Serfas 2004). Thus, transplantation
experiments in early pupae have shown that both of the
forewing eyespots of B. anynana are formed around groups
of organizing cells called foci; grafting a focus to a novel
position on the wing surface results in formation of an
ectopic eyespot around the grafted cell tissue (French and
Brakefield 1995). The foci are established in late larvae,
and then in early pupae, each focus sets up an information
gradient in the surrounding epithelial cells, presumably
via one or more diffusible signaling morphogens. These
cells then respond to the gradient and thus, depending on
their location relative to the signal source, become fated
to synthesize one of a series of different color pigments
just before adult eclosion.
Each of the seven eyespots on the ventral surface of the
hindwing of B. anynana expresses the same developmental
genes, including Notch, Distal-less, engrailed, and spalt, at
the same stages in eyespot formation (Brakefield et al. 1996;
Brunetti et al. 2001; Beldade et al. 2002a, 2005; Reed and
Serfas 2004). We have also established a series of spontaneous eyespot mutants of B. anynana. The mutant alleles
typically affect all the eyespots on a particular wing surface.
Furthermore, artificial selection experiments that were targeted on a particular trait (e.g., size or color composition)
of a single eyespot consistently produced highly correlated
responses in the other eyespots, especially on the same wing.
Taken together, these observations show that the different
eyespots are based on a common developmental pathway
and that many eyespot genes or alleles influence all the
repeated eyespot elements. This led us to design experiments
to explore the consequences of the shared genetical and
developmental mechanisms and to examine the potential
flexibility of different eyespots to evolve in different directions of morphological space (Brakefield 1998).
Exploring Evolutionary Constraints
S7
Figure 1: Occupancy of morphological space for the relative size of the two dorsal forewing eyespots. The four images of the wing are representative
examples of the wing pattern after 25 generations of artificial selection in Bicyclus anynana (from Beldade et al. 2002a) placed in roughly the correct
position in the depicted trait space; the star shows the average wild-type pattern for this species that formed the starting point for the selection in
each of the four directions. Circles show the positions of the mean patterns for different species of the genera Bicyclus (filled circles) and Mycalesis
(open circles) derived from measurements of specimens in museum collections. The dashed square encloses species for which both eyespots are very
small or absent and frequently difficult to measure. For species of Bicyclus and Mycalesis, total sample sizes were 1,371 and 575, respectively.
Artificial Selection Experiments
The wild-type wing pattern of the dorsal forewing of Bicyclus anynana consists of a small anterior eyespot and a
large posterior eyespot. The wing cells within which these
eyespots lie also differ in size in the same direction as the
eyespots. This pattern can be considered to be somewhere
in the middle of trait space for this pair of eyespots. Our
laboratory stock of B. anynana was established from more
than 80 gravid females collected at a single locality in
Malawi and was cultured to maintain high levels of standing genetic variation. An early selection experiment on the
size of the posterior eyespot revealed a moderate realized
heritability with rapid upward and downward responses
to selection (Monteiro et al. 1994). After only five generations of selection, both eyespots were substantially
larger in a high line and smaller in a low line, thus demonstrating a positive genetic correlation. The color composition of the eyespots changed very little, indicating low
genetic correlations between the two eyespot traits of size
and color (see also Monteiro et al. 1997a). Transplantation
experiments on early pupae involving the organizing foci
of both lines traced the phenotypic difference in eyespot
size mainly to differences in the activity of the signaling
cells of the eyespot foci. There was some evidence for
differences at the level of the threshold responses of the
epithelial cells to the focal signals, but foci donated by
pupae of the high line consistently yielded larger ectopic
eyespots in adults, whether grafted into pupal hosts of the
high or the low line (Monteiro et al. 1994).
A second experiment was then designed to explore
whether a phenotype in which one eyespot was smaller and
the other larger could be as readily produced by artificial
selection as the phenotypes in which both eyespots were
either small or large. Replicated lines were established from
the same founder population and selected toward each of
the four corners of trait space (see fig. 1), that is, along both
directions of the coupled axis, following the shared genetics
and development, and along both directions of the uncoupled axis, against the proposed genetic line of least resistance and plane of developmental bias. Selection took
place over 25 generations, although this included a period
of relaxed selection (Beldade et al. 2002b).
As expected, selection in both directions along the coupled axis produced rapid responses, with butterflies after
25 generations of selection having either no eyespots at
all or two very large eyespots, phenotypes completely dif-
S8 The American Naturalist
ferent from any present in the unselected population.
However, the populations along the other uncoupled axis
also responded in a dramatic way to selection, eventually
producing phenotypes in which one eyespot was very large
and the other absent or very small (see fig. 1). We concluded that this pattern in relative size of the two eyespots
behaved in a highly flexible manner, with no evidence of
any generative constraints (Beldade et al. 2002b). However,
this does not necessarily mean that there is no developmental bias or effect of the genetic correlation between
the two eyespots on patterns of diversity among different
taxa. An initial examination of photographs in a monograph on the genus Bicyclus (Condamin 1973; Roskam and
Brakefield 1999) suggested that although species occurred
with a large anterior eyespot and no posterior eyespot,
there were none showing the reversed pattern (Beldade et
al. 2002b). We have now begun to investigate the occupancy of this morphological space more carefully by using
measurements of specimens from museum collections not
only for 69 species of Bicyclus from sub-Saharan Africa
but also for 40 species of the closely related genus Mycalesis
that extends throughout Asia into Australia. Representative dorsal wing surfaces of four species of each genus are
shown in figure 2.
Comparisons of Eyespot Patterns among Species
The morphometric data for species of Bicyclus confirm the
occurrence of a rather wide range of variation among species for the relative size of the two eyespots on the dorsal
forewing. The pattern of occupation of morphological
space within the genus can then be compared with the
results of the artificial selection based on the standing
genetic variation at a single locality for one of the species
(fig. 1). A complete analysis of the data on the size of both
dorsal and ventral eyespots using a molecular phylogeny
for species of Bicyclus (Monteiro and Pierce 2001) will be
published elsewhere (J. C. Roskam, K. van der Linde, and
P. M. Brakefield, unpublished manuscript). Many species
of Bicyclus, including Bicyclus anynana, clearly lie along
the coupled axis that represents a positive genetic correlation among the two eyespots together with a common
developmental pathway. There are, however, species that
extend toward having a large anterior eyespot but a small
Figure 2: Representative dorsal surfaces of the right forewing and hindwing of (A) four species of Bicyclus and (B) four species of Mycalesis.
Note among species in each genus the variability in the size of the anterior
and posterior eyespots as well as in the patterns of relative eyespot size.
Species names in clockwise order from top left in each group are (A)
taenias, danckelmani, smithi, and simulacris and (B) sirius, splendens,
mineus, and oculus.
Exploring Evolutionary Constraints
posterior eyespot. The rest of the morphological space, the
region covering the opposite pattern not only in relative
size but also in the direction of both eyespots being very
large, is not occupied by any of the surveyed (extant)
species. Because artificial selection on a single laboratory
population has yielded replicate lines that have readily
crossed this morphological space (see fig. 1, superimposed
wings), we must conclude that such patterns are absent in
extant present-day taxa because of the way in which natural selection has operated in the past rather than because
of any strong generative constraints. Thus, while there is
clustering of species of Bicyclus around the axis of genetic
and developmental coupling that appears to reflect a genetic line of least resistance, in Schluter’s (1996) terminology, the gross pattern of occupancy must be accounted
for by environments found in nature in combination with
the legacies of natural selection.
This interpretation is given more weight by a comparison with the closely related genus Mycalesis (fig. 1). Here
there is a (weaker) trend toward following a parallel genetic
line of least resistance, but many species are then displaced
toward the pattern with a large posterior eyespot and a
small anterior eyespot (fig. 1, bottom right) rather than the
opposite pattern, as in Bicyclus (fig. 1, top left). Again,
considering all of this in the context of the results of artificial selection in B. anynana suggests that there is some
factor in terms of the environment and selection that has
influenced the different patterns of occupancy of morphological space shown by the two genera. Some recent
experimental work suggests that these eyespots are involved in sexual selection and mate choice (Breuker and
Brakefield 2002; Robertson and Monteiro 2005).
During courtship in B. anynana, the wings of males are
opened and closed very rapidly during a specific flickering
phase. The precise positioning of the male relative to the
female at this stage of courtship suggests that at least the
anterior eyespot on the apical tip of the forewing is then
visible to the female. Furthermore, preliminary data from
mate competition experiments (see Joron and Brakefield
2003) using free-flying males from the selected lines of
Beldade et al. (2002b) suggest that males with the larger
anterior eyespots have a higher mating success than those
in which this eyespot is small, while the posterior eyespot
has no affect on fitness (J. de Visser and P. M. Brakefield,
unpublished data). These observations need to be followed
up in more detail, but they may indicate some fundamental
difference in the structure of courtship between the two
genera that accounts for the striking difference in occupancy of morphological space (fig. 1). Whatever the details
of environment and selective history, the results to date
strongly suggest that the explanation for the difference
between the two genera is unlikely to have any substantial
developmental component. There may be relative con-
S9
straints, in the sense of a genetic line of least resistance,
that are sufficient to account for some clustering of species
along an axis of coupling for the two eyespots. However,
the high degree of flexibility found in the experiments with
25 generations of artificial selection in B. anynana indicates
that any such generative constraint is likely to have very
weak consequences, especially if natural selection is prolonged and strong.
The Potential for Generative Constraints
for Other Eyespot Traits
We have also examined the evolutionary and developmental genetics of an additional trait for the posterior
forewing eyespot, namely, color composition. Artificial selection yielded either gold butterflies, in which this eyespot
had a broad outer gold ring, or black butterflies with a
narrow gold ring (Monteiro et al. 1997a, 1997b). The estimate for realized heritability of eyespot color was similar
to that for size, and again the other dorsal eyespots showed
highly coordinated responses. There is also little, if any,
evidence for correlated responses for the two traits, size
and color (see also Beldade et al. 2002c; Beldade and Brakefield 2003a). Although these observations might suggest
comparable predictions for evolutionary responses to a
given intensity of natural selection, caution is needed because the two traits have a different developmental basis.
Thus, while the transplant experiments using pupae from
the selected lines with divergent eyespot size showed that
large eyespots result primarily from strong focal signals,
comparable trials using the gold and black lines traced no
effect to the focus but rather traced effects only to the
threshold responses to the focal signal of the host cells
that surround the graft and eventually produce the color
rings (see Beldade and Brakefield 2002). These observations can be interpreted as revealing two divergent developmental aspects of these traits: (1) that eyespot color is
more of a wing-level property (threshold responses of the
whole epithelial cell layer), while size is dependent on the
localized focal signal in the wing cell between each pair
of wing veins, and (2) that because size is to some degree
also influenced by the response thresholds, there are more
developmental options for changing size than color. What
are the evolutionary consequences of such differences, and
how did such differences in evolvability arise?
Figure 3 illustrates the ventral wing surfaces of four
representative species of each genus, Bicyclus and Mycalesis.
These wings tend to have more complete series of the
marginal eyespots. In each genus there are clearly species
characterized by eyespots with narrow gold rings and others characterized by broader gold rings. However, it appears that a mixture of eyespots that span a substantial
proportion of this overall range in color composition is
S10 The American Naturalist
not found in any of the species, especially within the surface of one of the wings. Thus, this trait contrasts sharply
with eyespot size, for which the pattern of relative size can
differ dramatically across species (fig. 2). Although such
patterns of variability in eyespot color among species remain to be properly quantified, they do suggest that it may
in some way be more difficult to uncouple the color composition of two eyespots on the same wing (i.e., one gold
and the other black) by artificial selection on the standing
genetic variation of a single species. Such experiments are
underway and appear to be yielding different results to
eyespot size (C. E. Allen, unpublished data).
Responses to Artificial Selection and Revealing
Variation in Development
It is worthwhile considering how, in general, the responses
observed in artificial selection experiments inform us
about the role of development in generating variation in
the phenotype that is relevant to natural selection and the
ecological arena. The answer is probably very little when
no further analysis is available. It is rather only when such
observations are combined with analyses in developmental
terms of what has happened—or, indeed, of what has not
happened—that relevant information will be forthcoming.
The phenotypes at the end of selection, whether in singleor in double-trait experiments, can be used to explore the
morphological changes in terms of the mechanisms of
development; they may indeed also reveal more about the
developmental process itself. Thus, follow-up studies can
identify which of the potential modes of variability in development or options for change have yielded the observed
changes in phenotype. Moreover, because artificial selection experiments usually target standing genetic variation
derived from one or more natural populations, they are
likely to provide more relevant information about the
(short-term) potential for phenotypic change in nature
than descriptions of variation based on mutagenic screens.
Thus, combining information from artificial selection
experiments about the developmental and genetical options for change in a complex morphology will inform us
about evolvability with respect to responses to short-term
selection in different directions of trait space. Furthermore,
matching experimental studies using a model species in
Figure 3: Representative ventral surfaces of the left wings of (A) four
species of Bicyclus and (B) four species of Mycalesis. Species names in
clockwise order from top left in each group are (A) simulacris, evadne,
taenias, and xeneas and (B) anaxias, mucia, duponcheli, and terminus.
Note the consistently narrow or broader outer gold rings of the marginal
eyespots within species in contrast to the variability among species in
each genus.
Exploring Evolutionary Constraints
S11
Figure 4: Diagram illustrating a scenario of the evolution of the eyespot pattern on the ventral hindwing in the genera Bicyclus and Mycalesis.
Evolution of the key innovation of making wing spots probably occurred in basal Lepidoptera. This was followed by a period of elaboration leading
to a modular pattern with serial repeats of similar eyespots having both larger size and color ring composition (below is a representative species
showing a comparable pattern extending over both hindwing and forewing, Mycalesis horsfeldi). For eyespot size, species in different selective
environments have diversified through a process of evolutionary tinkering. This has led to a high evolvability of the whole eyespot module and an
absence of any strong generative constraints. In contrast, the pattern of eyespot color composition apparently still shows low evolvability and the
presence of (strong) relative constraints.
the laboratory with a more comparative descriptive approach for the whole lineage will reveal the extent to which
the responses in short-term selection experiments can inform us about patterns of evolutionary change over long
timescales. To us, the most exciting prospect of this type
of integrative approach for amenable systems will be the
ability to compare patterns of evolvability across different
sets of traits involved in the evolution of complex morphologies. This will, in turn, provide more general insights
about how the properties of change in developmental processes contribute to observed patterns of evolution and
the occupancy of morphological space.
However, more of a challenge than making such descriptions of patterns of change in developmental pathways will
be disentangling cause and effect. Why is there more standing genetic and developmental variation along certain axes
of morphological variation than along others? Putting it
another way, why do genetic covariances among developmentally related traits have the properties they do? Is this
because of some fundamental ways in which the development processes work, or, rather, does it reflect the legacy of
how natural selection has influenced the morphologies concerned? Such questions will be answered only when an integrated approach is taken in certain key systems that lend
themselves to studies at different levels of biological organization, including how natural selection works in the eco-
logical arena. Again, this can be illustrated by referring to
the evolution in patterns of butterfly eyespots.
Perspectives: Fusing Micro- and Macroscales
of Evolutionary Change
Developmental studies on Bicyclus eyespots have already
suggested differences in the mechanisms of pattern determination for eyespot size and color composition (Monteiro et al. 1994, 1997a). Artificial selection experiments
in which two eyespots on the same wing surface have been
the target of selection are starting to reveal differences in
the evolvability of the pattern of two or more eyespots for
these different traits that may be traced back to the properties of eyespot development. If the early results from a
combination of the artificial selection experiments and
morphometric analyses across extant species are confirmed, a relative lack of genetic variation to facilitate an
uncoupling response for eyespot color in contrast to size
could be accounted for by (1) the absence of history of
selection in different environments for butterflies with a
combination of some eyespots with narrow and others
with broad rings, (2) a more absolute developmental constraint for eyespot color, perhaps relating to the wing-level
property of threshold responses to focal signals of different
eyespots, or (3) a combination of these two effects working
S12 The American Naturalist
in harness to make any substantial evolution of evolvability
in color less likely.
Figure 4 illustrates these scenarios. We argue that early
in the radiation of the Lepidoptera, an evolutionary novelty
that involved co-option of the hedgehog signaling pathway,
perhaps in a new functional context, as well as novel tissue,
late in development yielded the pattern of serial repeats of
small undifferentiated spots (see Brunetti et al. 2001). Such
patterns are found in many Lepidoptera, including basal
groups. There followed a period of evolutionary elaboration
with further co-opting and evolution of gene regulation to
yield the module of repeated structures, each with a potentially larger and more structured color pattern element. This
pattern is illustrated in the central column of figure 4; the
species Mycalesis horsfeldi represents a striking candidate for
possession of an eyespot pattern closely similar to such a
prototype module. Finally, species of Bicyclus and Mycalesis
in different environments of predator pressure, of light and
resting backgrounds, and of communities of conspecifics
evolved the ability for independent evolution in the serial
eyespot elements, at least for eyespot size. For some reason,
perhaps accounted for by a combination of selection history
and developmental bias, this process is less advanced for
eyespot color composition.
The important point here is that an integration of experimental work using a model species that is amenable
to a broad evo-devo framework with a more comparative
approach among many species can open up issues of the
roles of generative constraints to analysis. Such an integrative evolutionary biology will also enable links to be
made from the processes of adaptive evolution to patterns
of diversity among species. In addition, the basis of differences in evolvability can be investigated and eventually
related to differences in histories of selection as well as in
the genetical variances and developmental mechanisms
that generate the phenotype.
Acknowledgments
We are indebted to the various museums that provided
J.C.R. with access to their collections. We would also like
to thank the whole community of researchers on Bicyclus
anynana for its support. D. Schluter provided very useful
comments on the manuscript. Some of the ideas discussed
here were elaborated on during discussions of a National
Center for Ecological Analysis and Synthesis working
group organized by N. Eldredge and J. Thompson, to
whom P.M.B. is most grateful. P.M.B. also thanks M.
McPeek for his invitation to contribute to his symposium.
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Symposium Editor: Mark A. McPeek